Stem cells for transplantation and manufacturing method therefor

ABSTRACT

It is intended to provide MSCs for transplantation that have an improved post-transplantation cell survival rate and engraftment rate and are highly safe with fewer adverse reactions, and a method for conveniently producing MSCs for transplantation having a high cell survival rate and engraftment rate. As means therefor, the present invention provides a stem cell for transplantation comprising an MSC capable of overexpressing IL-10.

RELATED APPLICATIONS

This application is a continuation application of patent applicationSer. No. 16/821,630, filed Mar. 17, 2020, which is a continuation ofpatent application Ser. No. 15/966,917, filed Apr. 30, 2018, which is acontinuation of patent application Ser. No. 14/892,474, filed Nov. 19,2015, which is a 371 application of International Application No.PCT/JP2014/063448, having an international filing date of May 21, 2014,which claims priority to Japanese Patent Application No. 2013-108408,filed May 22, 2013, contents of which are incorporated herein byreference in their entirety.

REFERENCE TO APPENDIX [CD ROM/SEQUENCE LISTING]

This application is being filed electronically via EFS-Web and includesan electronically submitted Sequence Listing in .xml format. The .xmlfile contains a sequence listing entitled “515005_5000010_Seq_Listing”created on Jan. 30, 2023 and is 18,177 bytes in size. The sequencelisting contained in this .xml file is part of the specification andhereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates to a mesenchymal stem cell fortransplantation having a high survival rate and engraftment rate in celltransplantation, a method for producing the mesenchymal stem cells fortransplantation, and an agent enhancing post-transplantation mesenchymalstem cell engraftment.

BACKGROUND ART

Mesenchymal stem cells (hereinafter, also abbreviated to “MSCs” in thepresent specification) are somatic stem cells having the ability todifferentiate into cells belonging to the mesenchyme. MSCs areconsidered as the most realistic platform for cell transplantationtherapy at the moment, on the grounds that, for example, these cells arecapable of actively growing and thus facilitate securing the number ofcells, are less likely to cause rejection at the time oftransplantation, and have low ethical barriers. MSCs are expected to beapplied to regenerative medicine such as the regeneration of mesenchymalconnective tissue (e.g., bones, blood vessels, and cardiac muscle) orthe central nervous system.

MSCs also have the advantages that the cells are applicable toinflammation control therapy for inflammatory diseases and are highlyeffective for autologous transplantation therapy, which introduces atherapeutic gene to patient's own MSCs (Non Patent Literature 1). SinceMSCs further have the property of accumulating at a site havinginflammation or tissue damage, or immunosuppressing ability, thetransplantation of bone marrow-derived MSCs is carried out at the sametime with bone marrow transplantation for the purpose of promoting theengraftment of hematopoietic stem cells (Non Patent Literature 2). Theimmunosuppressive effect of MSCs is presumed to be limited to a localarea at which MSCs have accumulated, so as not to cause strong systemicimmunosuppression. MSCs are therefore considered to have higher safetythan that of immunosuppressants. Thus, their clinical effects areexpected.

Hence, clinical trials have been conducted so far on celltransplantation therapy which involves transplanting MSCs to targettissue, or inflammation control therapy for inflammatory diseases suchas graft versus host disease using the immunological control functionsof MSCs (Non Patent Literatures 3 to 6). The efficacy or safety of MSCshas been established in Canada and New Zealand where MSC drugs havealready been approved.

Nonetheless, MSCs present major problems: the cells have an unstablepost-transplantation survival rate or engraftment rate and thereforetend to result in graft failure; and their original properties aredifficult to maintain over a long period. Hence, the previous autologoustransplantation therapy using MSCs has failed to stably express atherapeutic gene.

CITATION LIST Non Patent Literature

-   Non Patent Literature 1: Connick et al., 2012, Lancet Neurol. 11    (2): 150-156-   Non Patent Literature 2: Carrancio S., et al., 2012, Cell    Transplant., 22: 1171-1183-   Non Patent Literature 3: M von Bonin et al., 2009, Bone Marrow    Transplant., 43: 245-251-   Non Patent Literature 4: Tolar et al., 2011, Hum. Gene Ther., 22:    257-262-   Non Patent Literature 5: Si Y. L., et al., 2011, Ageing Res. Rev.,    10: 93-103-   Non Patent Literature 6: Wang et al., 2012, J. Hematol. Oncol., 5:    19

SUMMARY OF INVENTION Technical Problem

In light of the problems mentioned above, an object of the presentinvention is to develop and provide MSCs that have an improvedpost-transplantation cell survival rate and engraftment rate and arehighly safe with fewer adverse reactions, and to provide a method forconveniently producing a stem cell for transplantation having a highcell survival rate and engraftment rate.

Solution to Problem

In order to attain the object, the present inventor has carried outvarious treatments to MSCs and consequently found that when ananti-inflammatory cytokine interleukin-10 (hereinafter, also abbreviatedto “IL-10” in the present specification) is overexpressed in MSCs, thepost-transplantation survival rate and engraftment rate of the MSCs aredrastically improved.

It has been further found that acquired immunological tolerance can beinduced by treating a recipient individual that undergoes celltransplantation or tissue transplantation, with MSCs together with animmunogen at least once before the transplantation.

The present invention is based on these findings and provides thefollowing:

(1) A stem cell for transplantation comprising an MSC capable ofoverexpressing IL-10.

(2) The stem cell for transplantation according to (1), wherein theoverexpression of IL-10 is caused by an exogenous IL-10 expressionsystem.

(3) The stem cell for transplantation according to (2), wherein theIL-10 expression system is a plasmid vector or a virus vector.

(4) The stem cell for transplantation according to any of (1) to (3),wherein the stem cell is intended for the regeneration of mesenchymalconnective tissue, the central nervous system, or the liver.

(5) A method for producing a stem cell for transplantation, comprisingthe step of introducing an IL-10 expression system capable ofoverexpressing IL-10 to an MSC.

(6) The method for producing a stem cell using transplantation accordingto (5), wherein the IL-10 expression system is a plasmid vector or avirus vector.

(7) An agent for enhancing engraftment of mesenchymal stem cell,comprising an IL-10 expression system capable of overexpressing IL-10 asan active ingredient.

(8) The agent for enhancing engraftment of mesenchymal stem cellaccording to (7), wherein the IL-10 expression system is a plasmidvector or a virus vector.

(9) A method for inducing acquired immunological tolerance, comprising:a first step of administering, within a period from 2 to 14 days beforeintroduction of an immunogen, an MSC and an immunogen having the sameimmunogenicity as that of said immunogen or a part thereof to arecipient individual at least once; and a second step of administeringMSCs to the recipient individual on the day before or the very day ofthe introduction of the immunogen.

(10) The method according to (9), wherein the MSC is a stem cell fortransplantation according to any of (1) to (4).

(11) The method according to (9) or (10), wherein the immunogen is avirus, a cell, a tissue, or an organ.

The present specification encompasses the contents described in thespecification and/or drawings of Japanese Patent Application No.2013-108408 on which the priority of the present application is based.

Advantageous Effects of Invention

According to the stem cell for transplantation of the present invention,MSCs that have a high post-transplantation cell survival rate andengraftment rate and are highly safe can be provided.

According to the method for producing a stem cell for transplantation ofthe present invention, a stem cell for transplantation having a highpost-transplantation engraftment rate can be conveniently produced.

According to the agent for enhancing of MSC of the present invention,the post-transplantation cell survival rate and engraftment rate of MSCscan be enhanced.

According to the method for inducing immunological tolerance of thepresent invention using MSCs, the post-transplantation rejection ofcells can be suppressed. Also, the post-transplantation cell survivalrate and engraftment rate can thereby be further enhanced.

DESCRIPTION OF DRAWINGS

FIGS. 1A-C show the survival rate of MSCs after recombinant IL-10 wasadministered by local injection together with MSCs to the lower leg ofeach NOD/Scid mouse. FIG. 1A shows the in vivo images of the mouse taken2 and 4 days after the administration. MSCs and recombinant IL-10(indicated by (+)) were administered to the left lower leg of the mouse,and only MSCs (indicated by (−)) were administered to the right lowerleg of the mouse. FIGS. 1B and 1C show quantitative values 2 days and 4days post-administration, respectively, calculated on the basis of theimages of FIG. 1A.

FIGS. 2A-B show the survival rate of MSCs after an IL-10 expression AAVvector and LacZ expression AAV vector as a control were eachadministered by local injection together with MSCs to the lower leg ofeach NOD/Scid mouse. FIG. 2A shows the in vivo image taken 9 days afterthe administration. The IL-10 expression AAV vector (IL-10) wasadministered to the left lower leg of the mouse, and the LacZ expressionAAV vector (LacZ) was administered to the right lower leg of the mouse.FIG. 2B shows a quantitative value calculated on the basis of the imageof FIG. 2A.

FIGS. 3A-C show the survival rate of MSCs after MSCs introducing anIL-10 expression plasmid DNA or a control GFP expression plasmid DNAwere administered by local injection to the lower leg of each NOD/Scidmouse. In this drawing, the MSCs transfected with IL-10 expressionplasmid DNA (IL-10(+)) was administered to the left lower leg of themouse, and the MSCs transfected with GFP expression plasmid DNA(IL-10(−)) was administered to the right lower leg of the mouse. FIG. 3Ashows the in vivo images of the mouse taken 3 days, 7 days, and 10 daysafter the administration (0 day). FIG. 3B shows an IL-10 expressionlevel in MSCs after culture for 12 days from the transfection of theIL-10 expression plasmid DNA or the GFP expression plasmid DNA. FIG. 3Cshows quantitative values 3 days, 7 days, 10 days, and 12 dayspost-administration calculated on the basis of the images of FIG. 3A.

FIG. 4 shows an IL-10 concentration in the serum of eachMSC-transplanted mouse. Cont represents a control mouse in which onlyMSCs were transplanted. pIL-10(+)MSCs represents two mice (No. 1 and No.2) in which IL-10(+)MSCs, which are the MSCs introducing a mouse IL-10expression plasmid DNA described in FIG. 3A, were transplanted.AAV1-IL-10 represents a mouse in which a mouse IL-10 expression AAVvector AAV1-IL-10 was administered together with MSCs to transplantationsite.

FIGS. 5A-B show the gene transfection of MSCs with an AAV vector and theexpression of IL-10. FIG. 5A shows results about MSCs 7 days after thegene transfection with a GFP expression AAV vector (AAV1-GFP). Thearrows represent MSCs expressing GFP by the transduction of the GFPexpression AAV vector. FIG. 5B shows results about the expression ofIL-10 in MSCs 7 days after the transduction of AAV1-GFP or a mouse IL-10expression AAV vector (AAV1-IL-10).

FIGS. 6A-B show the survival rate of MSCs after the stem cell fortransplantation of the present invention was administered by localinjection to each NOD/Scid mouse. FIG. 6A shows the in vivo images ofthe mouse the indicated numbers of days after the administration ofMSCs. In this drawing, vIL-10(+)MSCs, which are MSCs transducingAAV1-IL-10, were administered to the left lower leg of the mouse(IL-10(+)), and vIL-10(−)MSCs, which are MSCs transducing controlAAV1-GFP, were administered to the right lower leg of the mouse(IL-10(−)). FIG. 6B shows quantitative values (n=5) calculated on thebasis of the results of FIG. 6A.

FIGS. 7A-C are diagrams showing the engraftment of transplanted MSCs inmouse muscular tissue. FIG. 7A shows the image of the engraftment ofvIL-10(−)MSCs in the muscular tissue. FIG. 7B shows the image of theengraftment of vIL-10(+)MSCs in the muscular tissue. FIG. 7C shows theengraftment rates of vIL-10(−)MSCs and vIL-10(+)MSCs calculated from theimages of histological staining.

FIG. 8 is a conceptual diagram showing the flow of the transplantationof vIL-10(+)MSCs to a dog.

FIG. 9 is a diagram showing the engraftment of vIL-10(+)MSCs in dogmuscular tissue 30 days after transplantation. The dark spots encircledby broken lines or indicated by arrows represent vIL-10(+)MSCs engraftedin the endomysium and the perimysium.

FIG. 10A shows the positions of 4 sites in MSC-transplanted right andleft tibialis anterior muscle tissues. TA-2 is the transplantation siteof MSCs. FIG. 10B shows the survival rate of MSCs at each site in theright and left tibialis anterior muscle tissues and an IL-10concentration in the tissues 30 days after the transplantation. Thesurvival rates of MSCs in the left tibialis anterior muscle(vIL-10(−)MSCs were transplanted) and the right tibialis anterior muscle(vIL-10(+)MSCs were transplanted) are shown in (a) and (b),respectively. The IL-10 concentration in the muscular tissue of eachsite in the left tibialis anterior muscle and the right tibialisanterior muscle is shown in (c) and (d), respectively.

FIGS. 11A-B show the engraftment and myofiber formation ofnon-differentiation-induced MSCs. FIG. 11A is the pathologic image ofMSCs in muscular tissue. The sites indicated by arrows encircled bybroken lines represent the engraftment of transplanted vIL-10(+)MSCs atinflammation sites. The sites indicated by arrowheads representmyofibers newly formed from the engrafted MSCs. In FIG. 11B, the brightcells encircled by a broken line represent that MSCs were fused withmyoblasts in vitro.

FIGS. 12A-C are diagrams showing the effect of inducing immunologicaltolerance by the method for inducing acquired immunological tolerance ofthe present invention. FIG. 12A shows a control dog (Cont-AAV) in whichonly an immunogen AAV9-Luc was locally administered to the tibialisanterior muscle tissue of a normal dog without the immunologicaltolerance induction treatment of the present invention. FIG. 12B showsan immunological tolerance-induced dog (MSCs+AAV) in which MSCs werelocally administered together with the immunogen AAV9-Luc to the samesite of a normal dog as above on the basis of the method for inducingacquired immunological tolerance of the present invention. FIG. 12Cshows a control dog (Cont-MSCs) in which only MSCs were locallyadministered to the same site of a normal dog as above without theadministration of the immunogen AAV9-Luc. The left diagrams of FIGS. 12Ato 12C show a timetable for the experiment for each dog. i.v. denotesintravenous administration, and i.m. denotes intramuscularadministration (local administration). The right diagrams thereof showthat the tibialis anterior muscle tissue was biopsied, and theexpression of a marker gene of luciferase derived from the immunogenAAV9 was detected using an anti-luciferase antibody. In the diagrams,the luciferase-expressing cells are indicated as bright cells. In thediagrams, the scale bar is 100 μm.

FIGS. 13A-C are diagrams showing the effect of inducing immunologicaltolerance by the method for inducing acquired immunological tolerance ofthe present invention using intravenous administration. FIG. 13A shows acontrol dog (Cont-AAV) in which only an immunogen AAV9-Luc wasintravenously injected to a normal dog without the immunologicaltolerance induction treatment of the present invention. FIG. 13B showsan immunological tolerance-induced dog (MSCs+AAV) in which MSCs wereintravenously injected together with the immunogen AAV9-Luc to a normaldog on the basis of the method for inducing acquired immunologicaltolerance of the present invention.

FIG. 13C shows an immunological tolerance-induced dog (DMD/MSCs+AAV) inwhich MSCs were intravenously injected together with an immunogenAAV9-μDys to a muscular dystrophy-affected dog (DMD) on the basis of themethod for inducing acquired immunological tolerance of the presentinvention. The left diagrams of FIGS. 13A to 13C show the timetable forthe experiment for each dog. i.v. denotes intravenous administration.The right diagrams thereof show that the temporal muscle was biopsied,and the expression of luciferase was detected using an anti-luciferaseantibody (FIGS. 13A and 13B) or the expression of microdystrophin wasdetected using an anti-dystrophin antibody (FIG. 13C). The white spotsseen in each diagram are nuclei. In the diagrams, the luciferase- ormicrodystrophin-expressing cells are indicated as bright cells. In thediagrams, the scale bar is 100 μm (FIGS. 13A and 13B) and 50 μm (FIG.13C).

FIG. 14 is a diagram showing results about the running times of variousdogs in a 15-meter running test. In the diagram, “pre” in the lower tierof the abscissa represents the point in time when a musculardystrophy-affected dog (DMD) was pretreated by the method for inducingacquired immunological tolerance of the present invention. Other numericvalues represent the length of time (week) that passed from thereference date at which AAV9-μDys was introduced. The numeric valueswithin the parentheses in the upper tier of the abscissa represent theweekly age of the muscular dystrophy-affected dog (DMD).

FIG. 15 is a diagram showing the pathological evaluation of eachmuscular dystrophy-affected dog. The ordinate shows a score of theGrading Score, and the abscissa shows the age (months old) of theaffected dog.

DESCRIPTION OF EMBODIMENTS 1. Stem Cell for Transplantation 1-1. Summaryand Definition

The first aspect of the present invention provides a stem cell fortransplantation.

The “stem cell for transplantation” refers to a stem cell intended forcell transplantation and is aimed at being transplanted to a particulartissue of a recipient individual (or a recipient) and engrafted at thetransplantation site and/or neighboring sites thereof so that the cellis allowed to differentiate properly in response to the ambientenvironment. The stem cell for transplantation of this aspect can bepreferably used, particularly, in tissue regeneration such as thereplacement or regeneration of mesenchymal connective tissue (e.g.,bones, blood vessels, and cardiac muscle), the central nervous system,or liver tissue. In the present specification, the “recipientindividual” is a vertebrate, preferably a bird or a mammal (including ahuman, a dog, a cat, a rabbit, a pig, cattle, sheep, a goat, a horse, amonkey, and a rodent), more preferably a human.

The “engraftment” generally means that a transplanted cell, tissue, ororgan becomes capable of exerting its original functions at thetransplantation site. In the present specification, an object to betransplanted is a stem cell. In this respect, the engraftment meansthat, particularly, a transplanted stem cell has the functions of timelyexpressing a predetermined gene at the transplantation site orneighboring sites thereof, etc., and differentiating properly inresponse to the ambient environment. The “engraftment rate” described inthe present specification refers to the ratio of engrafted stem cells totransplanted stem cells. On the other hand, the “survival rate”described in the present specification refers to the ratio of stem cellssurviving after transplantation to transplanted stem cells. Theengraftment rate or the survival rate may be calculated from an absolutevalue such as the number of cells or may be calculated from a relativevalue such as cell-derived label intensity.

1-2. Constitution

The stem cell for transplantation of the present invention comprises amesenchymal stem cell capable of intracellularly overexpressinginterleukin-10.

The “mesenchymal stem cells (MSCs)” are, as mentioned above, cellshaving the ability to differentiate into cells belonging to themesenchyme, such as osteoblasts, adipocytes, myocytes, and chondrocytes.MSCs are also known to have the ability to differentiate plasticallyinto nerve cells (ectoderm-derived) or hepatic cells (endoderm-derived),across germ layers. The stem cell for transplantation of this aspect isbased on an MSC.

The “interleukin-10 (IL-10)” is an anti-inflammatory cytokine. IL-10 isproduced in many cells including type 2 helper T cells (Th2 cells) aswell as monocytes, macrophages, mast cells, activated B cells, andkeratinocytes, and is known to suppressively control the functions ofmacrophages in addition to suppressing inflammatory symptoms bysuppressively controlling inflammatory cytokine production or the likethrough its action on monocyte lineage cells.

In the present specification, the IL-10 encompasses wild-type IL-10 aswell as variant-type IL-10 and IL-10 fragments that maintain biologicalactivity equivalent to or higher than that of wild-type IL-10.

In the present specification, the “wild-type IL-10” refers to the IL-10protein of each organism species that is encoded by the most abundantallele among naturally occurring allele groups and has the originalfunctions of IL-10. The wild-type IL-10 corresponds to, for example,human IL-10 shown in SEQ ID NO: 1, mouse IL-10 shown in SEQ ID NO: 3,rat IL-10 shown in SEQ ID NO: 5, dog IL-10 shown in SEQ ID NO: 7,chimpanzee IL-10 shown in SEQ ID NO: 9, and bovine IL-10 shown in SEQ IDNO: 11.

In the present specification, the “variant-type IL-10” refers to anIL-10 protein having a variation in the wild-type IL-10. Specifically,the variant-type IL-10 means a variant containing the deletion,substitution, or addition of one or several amino acids in the aminoacid sequence of wild-type IL-10, or a variant having 85% or more or 90%or more, preferably 95% or more, 97% or more, 98% or more, or 99% ormore amino acid identity to the amino acid sequence. In this context,the term “several” refers to 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 or 3.The % “identity” refers to the ratio of identical amino acids of anamino acid sequence of interest to the total number of amino acids ofwild-type IL-10 when the amino acid sequence of wild-type IL-10 and theamino acid sequence of interest are aligned such that the maximum degreeof identity is achieved with or without optionally introduced gaps usinga protein search system based on BLAST or FASTA.

In the present specification, the “IL-10 fragment” refers to apolypeptide fragment of the wild-type IL-10 or the variant-type IL-10.The length and region of amino acids of the IL-10 fragment are notparticularly limited as long as the length and region allow thebiological activity of the wild-type IL-10 to be maintained. Usually, apolypeptide fragment containing the functional domain of IL-10 withoutdisruption is preferably used. In this context, the functional domainrefers to a region that is responsible for suppressive activity andfunctions specific for IL-10 or is essential for exerting the functions.

MSCs have an endogenous IL-10 gene encoding IL-10 and are capable ofexpressing IL-10 attributed to the gene. Hence, a feature of the stemcell for transplantation of this aspect is that the stem celloverexpresses IL-10 more than normal MSCs. In the present specification,the phrase “overexpressing IL-10” refers to an expression level 2 timesor more, preferably 5 times or more, more preferably 10 times or more,further preferably 20 times or more the expression level of IL-10 innormal MSCs.

The mechanism under which the stem cell for transplantation of thisaspect overexpresses IL-10 is not particularly limited. For example, theoverexpression may be based on the enhanced expression of endogenousIL-10 or may be based on an exogenous IL-10 expression system.

The “enhanced expression of endogenous IL-10” is not particularlylimited by its mechanism as long as the expression level of the IL-10gene on the genomes of MSCs is eventually enhanced. Examples of MSCshaving such enhanced expression of endogenous IL-10 include variant-typeMSCs allowed to overexpress IL-10 due to a variation in an enhancer or apromoter controlling the expression of the IL-10 gene, and variant-typeMSCs having, on their genomes, multicopy IL-10 genes resulting fromduplication.

The “exogenous IL-10 expression system” refers to a foreign IL-10expression system introduced in MSCs.

The “IL-10 expression system” refers to one expression unit containingthe IL-10 gene in an expressible state in MSCs. An expression unit thatpermits persistent expression of the IL-10 gene is preferred.

The “IL-10 gene” refers to a gene encoding the IL-10, or a fragmentthereof. The IL-10 gene corresponds to, for example, a human IL-10 geneshown in SEQ ID NO: 2 encoding the human IL-10 shown in SEQ ID NO: 1, amouse IL-10 gene shown in SEQ ID NO: 4 encoding the mouse IL-10 shown inSEQ ID NO: 3, a rat IL-10 gene shown in SEQ ID NO: 6 encoding the ratIL-10 shown in SEQ ID NO: 5, a dog IL-10 gene shown in SEQ ID NO: 8encoding the dog IL-10 shown in SEQ ID NO: 7, a chimpanzee IL-10 geneshown in SEQ ID NO: 10 encoding the chimpanzee IL-10 shown in SEQ ID NO:9, and a bovine IL-10 gene shown in SEQ ID NO: 12 encoding the bovineIL-10 shown in SEQ ID NO: 11.

The IL-10 expression system has the IL-10 gene as well as constituentsnecessary for expressing the IL-10 gene in MSCs. Such constituentsinclude, for example, a promoter and a terminator. In the IL-10expression system, the IL-10 gene is located in an expressible stateunder the control of the promoter and the terminator in a nucleic acidexpression system. The IL-10 expression system may additionally containan enhancer, a poly-A addition signal, a 5′-UTR (untranslated region)sequence, a tag or selective marker gene, a multicloning site, areplication origin, and the like.

The promoter is not particularly limited as long as the promoter isoperable in MSCs. For example, when MSCs containing the IL-10 expressionsystem are derived from a mammal, a promoter of a virus (e.g., humancytomegalovirus, retrovirus, polyomavirus, adenovirus, and simian virus40 (SV40)) or a mammalian cell-derived promoter such as elongationfactor 1α (EF1α) can be used, which is a promoter operable in a mammal.Promoters are known to be classified according to their expressioncontrol properties into an overexpression-type promoter, a constitutivepromoter, a site-specific promoter, a stage-specific promoter, or aninducible promoter, etc. The promoter for use in the IL-10 expressionsystem is preferably an overexpression-type promoter or a constitutivepromoter. It is desirable that, even if the expression of the endogenousIL-10 gene in transplanted stem cells is positionally or quantitativelycontrolled at the transplantation site, the IL-10 expression systemshould be capable of constantly expressing the IL-10 gene independentlyfrom the control. In the IL-10 expression system, the promoter ispositioned upstream from the start codon of the IL-10 gene.

The terminator is not particularly limited as long as its sequence canterminate the transcription of the IL-10 gene transcribed by thepromoter in MSCs. In the IL-10 expression system, the terminator ispositioned downstream from the stop codon of the IL-10 gene.

The IL-10 expression system includes an expression system prepared byisolating one expression unit necessary for expressing the IL-10 gene,as it is from the genome and incorporating the expression unit therein,and an expression system artificially constructed by combining arecombinant IL-10 gene with each constituent. In the present invention,any nucleic acid expression system can be used. The IL-10 expressionsystem may be a monocistronic system containing one IL-10 gene in onesystem or may be a polycistronic system containing two or more IL-10genes in one system.

Examples of the IL-10 expression system include an IL-10 expression-typeplasmid vector, an IL-10 expression-type virus vector, and an IL-10expression-type artificial chromosome vector. A virus vector ispreferred.

The “plasmid vector” refers to a gene vector modified from a plasmid.The plasmid moiety for use in the IL-10 expression-type plasmid vectoris not particularly limited as long as the IL-10 gene can be expressedin MSCs. The plasmid vector lacks the ability to self-renew in MSCs. Inthe case of using the plasmid vector, the expression of the IL-10 geneis therefore usually transient unless the plasmid vector is insertedinto the genomes of MSCs. A commercially available expression plasmid,for example, an expression plasmid for mammal cells, may be used as theplasmid moiety serving as the backbone of the IL-10 expression system.

The “virus vector” refers to a gene vector that exploits the infectiousability or the ability to replicate of a virus. The virus vector isconstituted by a virus particle containing a virus nucleic acid(including DNA or RNA) in which a foreign gene (here, the IL-10 gene) isincorporated after removal of genes related to pathogenicity from thevirus genome. In the IL-10 expression system, a vector derived from anyvirus known in the art can be used. Examples thereof include retrovirus(including lentivirus and mouse Moloney leukemia virus), adenovirus,adeno-associated virus (hereinafter, referred to as “AAV”), and Sendaivirus. AAV, which is a nonpathogenic virus belonging to the parvovirus,cannot grow autonomously due to deficiency in the ability to self-renewand is low infective because its growth requires the coinfection ofadenovirus or Herpes virus. This virus is also low immunogenic in hosts.Hence, the virus has the advantage that it is highly safe as a genevector. AAV has a wide host range and is capable of infecting variouscells such as myocytes, nerve cells, and hepatic cells. Thus, AAV isalso preferred as a virus vector for the IL-10 expression systemaccording to this aspect. The virus vector can be prepared as a virusparticle that is contained in a coat protein composed of a capsid or acoated particle and has the ability to infect cells.

The “artificial chromosome” includes human artificial chromosome (HAC).

The stem cell for transplantation of this aspect can contain a pluralityof identical or different IL-10 expression systems in one MSC. Thisincreases the number of IL-10 expression systems per cell, therebyincreasing an IL-10 expression level per MSC to attain an overexpressionstate, even if the expression level of IL-10 from each individual systemis low.

1-3. Effect

According to the stem cell for transplantation of this aspect, stemcells having a high post-transplantation cell survival rate andengraftment rate and fewer adverse reactions can be provided.Accordingly, the stem cell for transplantation of this aspect istransplanted in mesenchymal connective tissue (e.g., bones, bloodvessels, and cardiac muscle), the central nervous system, or the liverso that this transplanted cell is engrafted in the transplantationtissue and induced to differentiate appropriately in response to theambient environment. The stem cell for transplantation of this aspectcan therefore be applied to regenerative medicine such as tissueregeneration.

2. Method for Producing Stem Cell for Transplantation 2-1. Summary

The second aspect of the present invention provides a method forproducing a stem cell for transplantation. A feature of the productionmethod of this aspect is that an IL-10 expression system is introducedto MSCs to produce MSCs capable of overexpressing IL-10.

2-2. Method

The production method of this aspect comprises an IL-10 expressionsystem introduction step. The IL-10 expression system introduction steprefers to the step of introducing at least one IL-10 expression systemcapable of overexpressing IL-10 to an MSC.

The constitution of the IL-10 expression system used in the IL-10expression system introduction step may be the same as that of the IL-10expression system described in the first aspect.

The IL-10 expression system can be prepared according to a method knownin the art, for example, a method described in Green & Sambrook,Molecular Cloning, 2012, Fourth Ed., Cold Spring Harbor LaboratoryPress.

Hereinafter, the preparation of a plasmid vector or a virus vector asthe IL-10 expression system will be described with reference to specificexamples. However, the preparation of the IL-10 expression system is notlimited to the following.

(1) Preparation of Plasmid Vector

IL-10 gene cloning is carried out. The species from which the IL-10 geneused is derived is not particularly limited as long as activity as IL-10is possessed in MSCs to which the IL-10 expression system is introduced.An IL-10 gene derived from the same organism species as that of MSCs forintroduction is preferred. For example, in the case of introducing theIL-10 expression system to human-derived MSCs, the IL-10 gene ispreferably the human IL-10 gene shown in SEQ ID NO: 2. The IL-10 genecloning can be carried out according to a method known in the art, forexample, a method described in Green & Sambrook (2012) (idem). Forexample, in the case of cloning the human IL-10 gene, an appropriateregion is selected from the nucleotide sequence represented by SEQ IDNO: 2, and an oligonucleotide having this nucleotide sequence ischemically synthesized. The chemical synthesis can exploit thecommissioned synthesis service of a life science manufacturer. Next, thehuman IL-10 gene is isolated from a human cDNA library with theoligonucleotide as a probe on the basis of a method known in the art,for example, a plaque hybridization method. For the detailed geneisolation method, see, for example, Green & Sambrook (2012) (idem). Thehuman cDNA library is commercially available from each life sciencemanufacture, and such a commercially available product may therefore beused. Alternatively, oligonucleotides serving as a primer pair may bechemically synthesized on the basis of the nucleotide sequencerepresented by SEQ ID NO: 2, and the human IL-10 gene of interest can beamplified by a nucleic acid amplification method such as PCR using theprimer pair and a human genomic DNA or cDNA library as a template. Forthe nucleic acid amplification, DNA polymerase, such as pfu polymerase,which has 3′-5′ exonuclease activity and has high fidelity, ispreferably used. The detailed conditions for the nucleic acidamplification, see, for example, a method described in Innis M. et al.(Ed.), 1990, Academic Press, PCR Protocols: A Guide to Methods andApplications. The isolated human IL-10 gene is inserted, if necessary,to an appropriate plasmid and cloned in a microbe such as E. coli. Then,the full-length nucleotide sequence is confirmed on the basis of a knowntechnique. In addition, a human IL-10 cDNA clone is commerciallyavailable and may therefore be purchased for use.

Subsequently, the cloned human IL-10 gene is integrated into apredetermined site of a plasmid vector for expression. These series ofgene manipulation techniques are techniques well known in the art. Forthe detailed method, see, for example, Green & Sambrook (2012) (idem).

(2) Preparation of Virus Vector

The basic operation can follow the method for the plasmid vectormentioned above. First, the virus genome is prepared by a method knownin the art and then inserted to an appropriate cloning vector (e.g., E.coli-derived pBI series, pPZP series, pSMA series, pUC series, pBRseries, and pBluescript series) to obtain a recombinant. Next, the IL-10gene described in the preceding paragraph (1) is inserted to apredetermined site in the virus genome contained in the recombinant,followed by cloning. Subsequently, the virus genome region, which is theIL-10 expression system, can be excised from the recombinant withrestriction enzymes. In this way, the virus vector of interest can beobtained.

The method for introducing the IL-10 expression system into MSCs, i.e.,the method for transforming MSCs, can employ any appropriate methodknown in the art.

For example, when the IL-10 expression system is a plasmid vector, awell-known method can be used, such as an electroporation method, acalcium phosphate method, a liposome method, a DEAE dextran method,microinjection, lipofection, or binding with a cell membrane-permeablepeptide. For these specific methods, see methods well known in the art,for example, methods described in Green & Sambrook (2012) (idem).Alternatively, the plasmid vector may be introduced to MSCs using acommercially available nucleic acid-introducing agent such asLipofectamine 2000 (Life Technologies Corporation).

When the IL-10 expression system is a virus vector, the virus vector canbe allowed to virally infect MSCs so that the virus vector is transducedinto the MSCs. For the infection method, see a method well known in theart, for example, a method described in Green & Sambrook (2012) (idem),depending on the type of the virus used in the vector.

In order to stably and persistently express IL-10 in MSCs, the IL-10expression system may be inserted into the genomes of MSCs viahomologous recombination.

2-3. Effect

According to the production method of this aspect, stem cells fortransplantation based on MSCs can be produced conveniently andrelatively inexpensively. This method achieves easy obtainment of MSCshaving a high post-transplantation engraftment rate.

3. Agent Enhancing Mesenchymal Stem Cell Engraftment 3-1. Summary

The third aspect of the present invention provides an agent forenhancing engraftment of mesenchymal stem cell (agent enhancing MSCengraftment). The agent enhancing MSC engraftment of this aspect is anagent for converting MSCs to stem cells for transplantation having ahigh cell survival rate and engraftment rate. A feature thereof is todrastically enhance the post-transplantation survival rate andengraftment rate of MSCs.

3-2. Constitution

The agent enhancing MSC engraftment of this aspect comprises an IL-10expression system capable of overexpressing IL-10 as an activeingredient. This IL-10 expression system has the same constitution asthat of the IL-10 expression system described in the first aspect, sothat the description thereof is omitted here. The agent enhancing MSCengraftment of this aspect may be in a dried solid state or may be in aliquid state in which the agent has been dissolved in an appropriatebuffer. Because the IL-10 expression system serving as an activeingredient is constituted by a nucleic acid, it is preferred that theIL-10 expression system should be stored under conditions free fromnuclease and where the IL-10 expression system is stably retainedwithout being degraded by the agent enhancing MSC engraftment, forexample, at a subzero temperature.

3-3. Use Method

The agent enhancing MSC engraftment of this aspect can attain itspurpose by introducing the IL-10 expression system serving as an activeingredient into MSCs, which are cells to be transplanted. The method forintroducing the IL-10 expression system into MSCs differs depending onthe type of the IL-10 expression system and can be basically carried outaccording to the method for introducing the IL-10 expression system intoMSCs (Method for transforming MSCs) described in the paragraph “2.Method for producing stem cell for transplantation”. The MSCs, which arecells to be transplanted, may not only be wild-type MSCs isolated froman organism and cultured, but may also be variant-type MSCs in which amarker gene such as a luciferase expression vector has been introducedin advance.

3-4. Effect

According to the agent enhancing MSC engraftment of this aspect, MSCsfor transplantation having a high cell survival rate and engraftmentrate can be easily prepared from normal MSCs.

4. Method for Inducing Acquired Immunological Tolerance 4-1. Summary

The fourth aspect of the present invention provides a method forinducing acquired immunological tolerance. A feature of the method ofthis aspect is to induce the immunological tolerance of a recipientindividual in an acquired manner so that immune response to animmunogen, such as a cell or a tissue, which is introduced into theorganism, is suppressed.

4-2. Method

The method of this aspect comprises a first step and a second step asessential steps.

(1) First Step

The “first step” is the step of administering, within a period of thepredetermined number of days before introduction of an immunogen, an MSCand the immunogen or a part thereof to a recipient individual.

The “immunogen” in this aspect refers to a substance havingimmunogenicity with the aim of being introduced into an organism and ismainly a protein. Examples thereof include viruses, cells, tissues, andorgans. The immunogen used in this step and the immunogen used in thesubsequent second step need only to have immunogenicity different fromthat of the recipient individual and are not necessarily required to bethe same types of immunogens. The phrase “having differentimmunogenicity” refers to differing in immunogenicity or antigenicity.The phrase “having different immunogenicity” refers to, for example,differing in human leucocyte antigen (HLA) type or virus serotype. Inthe case of transplanting a tissue derived from a human different from arecipient individual to a human as the recipient individual (recipientperson), the transplanted tissue and the recipient person differ in HLAtype, which is immunogenicity, from each other.

The term “a part thereof” in this aspect means a moiety of the immunogenhaving a size that permits local administration or intravascularadministration to the recipient individual, when the immunogen to betransplanted has too large a size to be locally administered orintravascularly administered via injection or the like. For example,when the immunogen is a particular tissue, the term “a part thereof”refers to one cell or a mass of several cells constituting the tissue.

The MSC used in this aspect may be a normal MSC or may be the MSC as thestem cell for transplantation capable of overexpressing IL-10 describedin the first aspect. In the case of using the stem cell fortransplantation as the MSC, only this MSC can be administered in thisstep.

In this step, the transplantation date of the immunogen to the recipientindividual is defined as a reference date (day 0), and the MSC and theimmunogen or a part thereof are administered into the recipientindividual within a period (13 days) from 2 to 14 days before thereference date, preferably within a period (9 days) from 2 to 10 daysbefore the reference date, more preferably within a period (7 days) from2 to 8 days before the reference date. The MSC and the immunogen or apart thereof may be administered to the recipient individual at the sametime, either after being mixed with each other and infecting the MSCwith the immunogen (in the case of a virus) or individually, or may beadministered sequentially. In this case, the order of administration isnot limited. The immunogen or a part thereof may be administered to therecipient individual after administration of MSCs, or vice versa. Theinterval of administration between them is within 30 minutes, preferablywithin 10 minutes.

The number of doses within the aforementioned period can be at leastone. Usually, one to several doses suffice. However, the administrationis carried out once per day as a rule. The administration timing withinthe period is not limited. For example, when the number of doses withinthe period is one, this administration is preferably carried out withina period from 6 to 8 days before the reference date.

The method for administration to the recipient individual is notlimited, and a local administration method which involves directlyadministering the immunogen to an intended transplantation site or theneighborhood thereof, or a systemic administration method mediated bythe circulatory system can be preferably used. Examples of the localadministration method include subcutaneous administration, intramuscularadministration, intraarticular administration, intramedullaryadministration, administration into tissues (including administrationinto organs), and intraperitoneal administration. Examples of thesystemic administration method mediated by the circulatory systeminclude intravenous administration, intraarterial administration, andintra-lymphatic administration. Any of these administration methods arepreferably based on the administration of a liquid formulation byinjection. The administration method based on intravenous injection isan administration method suitable for this aspect.

The number of MSCs and the amount of the immunogen per dose in this stepare not limited. The number of MSCs can fall within the range of, forexample, 5×10⁵ cells/kg B.W. (Body Weight) to 2×10⁶ cells/kg B.W. Theamount of the immunogen differs depending on the type thereof and canfall within the range of 1×10⁵ v.g./cell to 1×10⁶ v.g./cell for a virusor the range of 1×10² to 1×10¹⁰ cells/kg B.W.

(2) Second Step

The “second step” refers to the step of administering an MSC to therecipient individual on the day before or the very day of theintroduction of the immunogen.

The MSC to be administered in this step may be an MSC different fromthat administered in the first step. For example, a normal MSC can beadministered in the first step, and the stem cell for transplantationcapable of overexpressing IL-10 described in the first aspect can beadministered in this step.

The MSC may be administered on any of the day before and the very day ofthe immunogen introduction date, i.e., the reference date, and ispreferably administered on the day before. In the case of administeringthe MSC on the very day of the reference date, the MSC may beadministered separately from the immunogen to be introduced, or may beadministered at the same time with the introduction of the immunogen.For the administration separate from the introduction of the immunogen,it is preferred to introduce the immunogen after administration of theMSC. For the administration at the same time with the introduction ofthe immunogen, the immunogen may be introduced as a mixture with theMSC, provided that the immunogen to be introduced can be locallyadministered or systemically administered via the circulatory system, aswith a virus.

4-3. Effect

In the recipient individual treated by the method for inducing acquiredimmunological tolerance of this aspect, immunological tolerance isinduced against the immunogen used in the pretreatment. Accordingly, theimmunogen can then be introduced to the recipient individual by a methodknown in the art, thereby suppressing immune response to the introducedimmunogen in the recipient individual. Hence, when the immunogen is acell, a tissue, or an organ, rejection caused by immune response in therecipient individual resulting from the transplantation thereof can besuppressed or circumvented. As a result, the post-transplantationsurvival rate of the cell or the like can be enhanced, and theengraftment rate thereof in the recipient individual can be enhanced.

Comparative Example 1 <Verification of Effect of Improving Survival Rateof MSCs by Recombinant IL-10>

(Objective)

The objective is to verify the effect of improving the survival rate oftransplanted cells after recombinant mouse IL-10 was introduced togetherwith MSCs into muscular tissue.

(Method)

1. Cultured Cells and Culture Conditions

A luciferase expression vector pLuc was introduced to rat bonemarrow-derived MSCs (SD-rat MSCs) to establish a cell line SD-ratMSCs-Luc stably expressing luciferase. SD-rat MSCs-Luc was inoculated toa dish containing a DMED/F-12 (1:1) (GIBCO) medium containing 10% FBS(Nichirei Corp.) and cultured at 37° C. in the presence of 5% CO₂.Hereinafter, the “MSCs” described in Comparative Examples 1 and 2 andExamples 1 and 2 in the present specification mean the “SD-rat MSCs-Luc”used in the experiment.

2. Local Transplantation to Mouse

The MSCs thus cultured were recovered by trypsin treatment, and thenumber of cells was adjusted with PBS into 5×10⁶ cells/100 μL to preparean MSC solution. Recombinant mouse IL-10 (PeproTech, Inc.) was adjustedto 0.3 μg/10 μL saline (hereinafter, abbreviated to “0.3 μg of IL-10”)and 1.0 μg/10 μL saline (hereinafter, abbreviated to “1.0 μg of IL-10”),and each solution was mixed with the MSC solution. The resulting mixturewas administered by local injection to the left lower leg of eachNOD/Scid mouse (3 months old, male) to transplant the MSCs. For acontrol, an MSC solution free from recombinant IL-10 (IL-10(−)MSCs) wasadministered by local injection to the right lower leg. 1 and 4 daysafter the administration, the same amount of recombinant mouse IL-10 asabove was additionally administered to the left lower leg.

3. Verification of Survival Rate of Transplanted MSCs

2, 4, and 9 days after the administration, in vivo bioimaging analysiswas conducted to measure the luciferase luminescence activity of thetransplanted MSCs ex vivo from the mouse. 200 to 300 μL of a 15 mg/mLluciferin solution was intraperitoneally administered at 150 mg/kg tothe mouse under 2% isoflurane anesthesia. 20 minutes later, theluciferase luminescence intensity was measured using IVIS® ImagingSystem (PerkinElmer, Inc.) and digitalized as ROI (region of interest)using software Living Image® 3.2. Then, the survival rate of thetransplanted MSCs after the administration was quantitatively analyzed.When the ROI of the control IL-10(−)MSCs was defined as 1, the ratio ofthe luminescence value of IL-10(+)MSCs was calculated.

(Results)

The results are shown in FIGS. 1A-C. FIG. 1A shows the images of themouse (n=2) taken 2 and 4 days after the administration. FIG. 1B showsquantitative values calculated on the basis of the images of FIG. 1A.When 0.3 μg of IL-10 was administered together with MSCs, no largedifference in survival rate was confirmed as compared with IL-10(−)MSCs,as to both the results 2 days and 4 days post-administration. Bycontrast, when 1.0 μg of IL-10 was administered together with MSCs, thesurvival rate was 3.2 times and 1.7 times 2 days and 4 days,respectively, after the administration. These results demonstrateddose-dependent improvement in the survival rate of transplanted MSCs byIL-10.

However, for both 0.3 μg and 1.0 μg of IL-10, the survival of MSCs wasunable to be confirmed 9 days after the administration. Thus, it wasrevealed that, in the case of administering IL-10 together with MSCsinto a tissue, the survival rate of the transplanted MSCs can beimproved in a manner dependent on the dose of IL-10, though this effectis transient and engraftment is difficult.

Comparative Example 2

<Verification of Effect of Improving Survival Rate of MSCs by ConcurrentAdministration of IL-10 Expression AAV Vector>

(Objective)

The objective is to verify the effect of improving the survival rate ofMSCs after an IL-10 expression AAV vector was administered at the sametime with the MSCs into muscular tissue.

(Method)

1. Preparation of Recombinant AAV

In this Comparative Example, recombinant AAV1 (rAAV1) was used. TherAAV1 was prepared according to the method of Okada et al. (Okada, T.,et al., 2009, Hum. Gene Ther., 20: 1013-1021; and Okada, T., et al.,2005, Hum. Gene Ther., 16: 1212-1218).

For the preparation of the IL-10 expression AAV vector(AAV1-CAG-mIL-10-WPRE(EW); hereinafter, referred to as “AAV1-IL-10”),first, RNA was collected from mouse peripheral blood lymphocytes andthen reverse-transcribed into cDNA by RT-PCR. The coding region of mouseIL-10 was amplified as a recombinant mouse IL-10 gene by PCR using thecDNA as a template and a primer set consisting of SEQ ID NO: 13 (Fw:5′-CGCGGATCCATGCCTGGCTCAGCACTGCTATGCT-3′) and 14 (Rv:5′-GAGGATCCTCTTAGCTTTTCATTTTGATCAT-3′) having a 5′-BamHI tag.Subsequently, the amplification product was cleaved with BamHI and thenintegrated into the BamHI site of a pW-CAG-WPRE vector (Okada, T., etal., 2005, Hum. Gene Ther., 16: 1212-1218) to prepare AAV1-IL-10. TheAAV1-IL-10 is capable of persistently expressing IL-10 in a tissue thathas received it.

On the other hand, a LacZ expression AAV vector (pAAV-LacZ; hereinafter,referred to as AAV1-LacZ) (Agilent Technologies, Inc.) containing LacZinstead of IL-10 was used as a control.

2. Local Transplantation to Mouse and Verification of Survival Rate ofMSCs

The basic method followed the method described in Comparative Example 1.4 days before administration, 100 μL of 10 μM cardiotoxin (Sigma-AldrichCorp.) was administered by local injection to the right and left lowerlegs of each NOD/Scid mouse (5 months old, male) to induce muscleinjury. AAV1-IL-10 and AAV1-LacZ were each adjusted to 5×10⁸ g.c. and1×10⁹ g.c. and mixed with an MSC solution having 5×10⁶ cells/100 μL.Each mixture was administered by local injection to the left lower legfor AAV1-IL-10 and the right lower leg for AAV1-LacZ, of the mouse. 9days after the administration, the survival rate of MSCs wasquantitatively analyzed by in vivo bioimaging analysis.

(Results)

The results are shown in FIGS. 2A-B. FIG. 2A shows the image of themouse taken 9 days after the administration. FIG. 2B shows aquantitative value (n=4) calculated on the basis of the results of FIG.2A. Even when AAV1-IL-10 was administered by local injection, the effectof improving the survival rate of the transplanted MSCs was notconfirmed, irrespective of the dose. Thus, it was revealed that MSCscannot be engrafted even if IL-10 is persistently expressed in a tissuethat has received it.

Example 1 <Verification of Effect of Improving Survival Rate of MSCs byMSCs Intracellularly Overexpressing IL-10>

(Objective)

The objective is to verify the effect of improving the survival rate ofMSCs after the intracellular expression level of IL-10 in MSCs wasincreased by use of an IL-10 expression plasmid, and an IL-10concentration in serum.

(Method)

1. Gene Transfection of MSCs with IL-10 Expression Plasmid DNA

2 μg of a mouse IL-10 expression plasmid DNA (pW-CAG-mIL-10-WPRE) or acontrol GFP expression plasmid DNA (pW-CAG-EGFP-WPRE) was mixed with 100μL of a Nucleofection solution (Human MSC Nucleofector kit, Lonza GroupLtd.)/5×10⁵ MSCs, and each mixture was used in gene transfection usingan Amaxa Nucleofector system (C-17 mode). The pW-CAG-mIL-10-WPRE wasprepared by introducing the recombinant mouse IL-10 gene prepared inComparative Example 2 to the BamHI site of pW-CAG-WPRE. ThepW-CAG-EGFP-WPRE was prepared by introducing an EGFP gene to the BamHIsite of pW-CAG-WPRE (Okada, T., et al., 2005, Hum. Gene Ther., 16:1212-1218). The detailed procedures followed the protocol attached tothis product. Hereinafter, the MSCs transfected with the mouse IL-10expression plasmid DNA are referred to as pIL-10(+)MSCs, and the MSCsintroducing the control GFP expression plasmid DNA are referred to aspIL-10(−)MSCs.

2. Verification of Administration by Local Injection to Mouse andSurvival Rate

The basic method followed the method described in Comparative Example 1.Immediately after the introduction of each gene to MSCs according to thepreceding paragraph “1. Gene transfection of MSCs with IL-10 expressionplasmid DNA”, a 7.5×10⁶ cells/100 μL pIL-10(+)MSCs solution and apIL-10(−) MSCs solution were administered by local injection at equaldoses to the left lower leg and the right lower leg, respectively, ofeach NOD/Scid mouse (6 months old, female). 3, 7, 10, and 13 days afterthe administration, the survival rate of MSCs was quantitativelyanalyzed by in vivo bioimaging analysis.

3. IL-10 Expression Analysis

For the confirmation of the expression of IL-10 derived from MSCs, apart of the MSCs introducing the gene in the preceding paragraph “1.Gene transfection of MSCs with IL-10 expression plasmid DNA” wasinoculated to the aforementioned DMED/F-12 (1:1) medium containing 10%FBS and cultured at 37° C. for 12 days in the presence of 5% CO₂, andthe resulting culture supernatant was used in ELISA (Mouse IL-10 ELISAKit, Thermo Fisher Scientific Inc.) according to the attached protocol.

4. Measurement of IL-10 Concentration in Serum

13 days after the administration of MSCs, blood was collected from theheart of the mouse, and the IL-10 concentration in the serum wasquantified using Mouse IL-10 ELISA Kit (Thermo Fisher Scientific Inc.).As a control, blood was similarly collected from the mouse in whichAAV1-IL-10 was administered by local injection to the MSCtransplantation site in Comparative Example 2, and the IL-10concentration in serum was quantified using this kit.

(Results)

The results are shown in FIGS. 3A-C. FIG. 3A shows the images of themouse taken the indicated numbers of days after the administration ofMSCs. FIG. 3B shows quantitative values (n=2) calculated on the basis ofthe results of FIG. 3A. FIG. 3C shows the expression level of IL-10derived from MSCs. pIL-10(+)MSCs exhibited values as high as 69 timesand 3.4 times 7 days and 10 days, respectively, after the administrationas compared with pIL-10(−)MSCs. By contrast, the signal intensity ofluciferase was unable to be confirmed 12 days after the administration.These results suggested that the overexpression of IL-10 in MSCsimproves the post-transplantation survival rate of the MSCs and therebypermits engraftment of the MSCs even 10 days after the administration,which is difficult to achieve by conventional methods.

FIG. 4 shows the IL-10 concentration in the serum of theMSC-transplanted mouse. The mouse in which AAV1-IL-10 was administeredby local injection to the MSC transplantation site in ComparativeExample 2 had a mild rise in the IL-10 concentration (up to 150 pg/mL)in serum. By contrast, two mice (No. 1 and No. 2) of this Example inwhich pIL-10(+)MSCs intracellularly expressing IL-10 were transplantedhad only a slight rise in IL-10 concentration in serum. In general, anelevated IL-10 concentration in serum causes adverse effects such ashematopoietic injury or reduction in immunity. Thus, it was demonstratedthat the transplantation of MSCs intracellularly overexpressing IL-10causes fewer adverse reactions resulting from an IL-10 concentration inserum and has high safety.

Example 2 <Verification of Effect of Improving Survival Rate andEngraftment Rate by MSCs Overexpressing IL-10>

(Objective)

The objective is to verify the effect of improving the survival rate andengraftment rate of MSCs after the intracellular expression level ofIL-10 in the MSCs was increased by use of an IL-10 expression AAVvector.

(Method)

1. Gene Transfection of MSCs with AAV Vector and Confirmation ofExpression

The recombinant AAV was prepared according to the method described inComparative Example 2. SD-rat MSCs-Luc was inoculated at 1×10⁵cells/well to a 24 well-plate (IWAKI/AGC Techno Glass Co., Ltd.)containing a DMED/F-12 (1:1) medium containing 10% FBS. After celladhesion, an unpurified culture supernatant containing the mouse IL-10expression AAV vector AAV1-IL-10 or a GFP expression AAV vectorAAV1-CAG-EGFP-WPRE(EW) (hereinafter, referred to as “AAV1-GFP”) at5.0×10¹⁰ g.c. was added thereto for the gene transfection of the SD-ratMSCs. After overnight culture at 37° C. in the presence of 5% CO₂, themedium was replaced with a fresh one every 2 days, and the culturesolution was recovered for 2 days from 5 to 7 days after the genetransduction. 7 days after the gene transfection, GFP-expressing cellswere observed under a fluorescence microscope (Olympus Corp., IX71) todetermine gene transduction efficiency. The IL-10 expression level inthe recovered culture supernatant was quantified by ELISA (Mouse IL-10ELISA Kit, Thermo Fisher Scientific Inc.). The AAV1-GFP was prepared byinserting the EGFP gene prepared in Example 1 to the BamHI site ofAAV1-CAG-WPRE(EW).

2. Administration by Local Injection to Mouse and Verification ofSurvival Rate and Engraftment Rate

The basic method followed the method described in Comparative Example 1.Similarly to the preceding paragraph “1. Gene transduction of MSCs withAAV vector and confirmation of expression”, the gene transduction ofMSCs was carried out using an unpurified culture supernatant containingthe AAV1-IL-10 or the control AAV1-GFP. A DMED/F-12 (1:1) mediumcontaining 10% FBS supplemented with each AAV vector at 6.0×10¹² g.c.was placed in a T225 flask (Thermo Fisher Scientific Inc.) to which1.6×10⁷ cells of MSCs adhered. After culture at 37° C. for 5 days in thepresence of 5% CO₂, each AAV vector was added again at 1.1×10¹³g.c./flask, and the culture was continued for 2 days for genetransduction. Hereinafter, the MSCs transducing the AAV1-IL-10, whichcorrespond to the stem cell for transplantation of the presentinvention, are referred to as “vIL-10(+)MSCs”, and the MSCs transducingthe control AAV1-GFP are referred to as “vIL-10(−)MSCs”. After the genetransfection, the MSCs (1.0×10⁷ cells) were administered by localinjection to the left lower leg (vIL-10(+)MSCs) and the right lower leg(vIL-10(−)MSCs) of each NOD/Scid mouse (3 months old, 5 females and 1male). 3, 7, 18, 27, 31, 34, 42, 49, 54, and 67 days after theadministration, the survival rate of MSCs was quantitatively analyzed byin vivo bioimaging analysis.

In order to confirm the engraftment of the transplanted MSCs, the mousewas dissected 74 days after the administration, and a frozen block ofthe MSC-transplanted muscular tissue was prepared andimmunohistologically stained. MSCs engrafted in the muscular tissue weredetected by DAB staining (VECTASTAIN Elite ABC, Vector Laboratories,Inc.) using an antibody against luciferase introduced in the MSCs(rabbit anti-firefly Luciferase antibody, 1/100 diluted, Abcam Plc.) andsubsequent H & E staining. The number of MSCs per unit area was countedto calculate an engraftment rate.

(Results)

1. Gene Transduction of MSCs with AAV Vector and Confirmation ofExpression

The results are shown in FIGS. 5A-B. FIG. 5A shows the results about thegene transduction of vIL-10(−)MSCs. FIG. 5B shows the results about theexpression of IL-10 in vIL-10(+)MSCs.

The gene transduction was confirmed on the basis of the number ofGFP-positive cells. As shown in FIG. 5A, many MSCs after the genetransduction were GFP-positive cells, demonstrating that the genetransduction of MSCs using the AAV vector is efficiency carried out. Asshown in FIG. 5B, IL-10 was able to be detected in the culturesupernatant of vIL-10(+)MSCs, showing that IL-10 can be highly expressedin vIL-10(+)MSCs.

2. Administration by Local Injection to Mouse and Verification ofSurvival Rate and Engraftment Rate

FIGS. 6A-B show the images of the mouse and the survival rate of MSCs.FIG. 6A shows the images of the mouse taken the indicated numbers ofdays after the administration of MSCs. FIG. 6B shows quantitative values(n=5) calculated on the basis of the results of FIG. 6A. The signalintensity of luciferase in vIL-10(+)MSCs 31 days after theadministration exhibited a value 9.7 times higher than that ofvIL-10(−)MSCs. Further surprisingly, vIL-10(+)MSCs maintained the highsignal of luciferase even 67 days after the administration. Thus,significant improvement in the survival rate of MSCs was able to beconfirmed. These results demonstrated that MSCs are allowed topersistently express IL-10 using the AAV vector, and can thereby surviveover a long period in a tissue in which the MSCs have been transplanted.

FIGS. 7A-C are diagrams showing the engraftment of MSCs in musculartissue, which was the transplantation site. FIGS. 7A and 7B show theimages of the engraftment of vIL-10(−)MSCs and vIL-10(+)MSCs,respectively, in the muscular tissue. FIG. 7C shows the engraftmentrates of vIL-10(−)MSCs and vIL-10(+)MSCs calculated by measuring thenumber of MSCs per unit area from the images of histological staining.

vIL-10(+)MSCs are indicated by dark spots encircled by broken lines inFIG. 7B by the DAB staining, demonstrating their engraftment in theendomysium and the perimysium. The results of FIG. 7C also demonstratedthat the vIL-10(+)MSCs, which correspond to the stem cell fortransplantation of the present invention, have engraftment efficiencyimproved by two or more times as compared with the control vIL-10(−)MSCswith a significant difference (p<0.005).

Example 3

<Verification of Improvement in Survival Rate and Engraftment Rate inExperiment of Transplantation of MSCs Transduced with IL-10 ExpressionAAV Vector into Dog>

(Objective)

The objective is to verify, in dogs, the effect of improving thesurvival and engraftment rates of AAV1-IL-10-introduced MSCs, which wasdemonstrated in mice.

(Method)

1. Preparation of Dog Bone Marrow-Derived MSCs

Donor and recipient dogs were selected from DLA (dog leukocyteantigen)-matched male and female pairs of beagles. As illustrated inFIG. 8 , 2 mL of a bone marrow fluid was collected from the fore-legright and left upper arms of the donor normal dog. The bone marrow fluidwas cultured in 2 mL of RPMI-1640 (Life Technologies Corporation)containing 20 U/mL heparin. Monocytes were isolated (1.3×10⁸ cells)under a density gradient using Histopaque-1077 (Sigma-Aldrich Corp.).Then, a CD271-positive fraction having the high ability to grow (MSCResearch Tool Box-CD271 (LNGFR) containing CD271 (LNGFR)-PE & Anti-PEMicro Beads (Miltenyi Biotec) was concentrated using immuno-magneticbeads (MACS® Columns and MACS Separators, Miltenyi Biotec)(CD271-positive cells=1.4 to 5×10⁶ cells). The detailed method followedthe attached protocol. The recovered cells were seeded to a 6-well plate(IWAKI/AGC Techno Glass Co., Ltd.) and cultured in nonhaematopoietic(NH) Expansion Medium (Miltenyi Biotec) supplemented with 100 U/mLpenicillin and 100 μg/mL streptomycin (Sigma-Aldrich Corp.). The mediumwas replaced with a fresh one every 3 days, and the cells were allowedto grow into a level corresponding to ten T225 flasks through 4passages. The detailed preparation followed Kasahara, Y., et al., 2012,Mol Ther., 20 (1): 168-77. CD271⁺ MSCs were infected with a luciferaseexpression lentivirus vector (200 μL) in the presence of Polybrene (8μg/mL) and cultured at 32° C. for 2 days in NH Expansion Medium (T225flask) for gene transfection. Subsequently, AAV1-GFP or AAV1-IL-10(8.0×10¹² g.c. each) was cultured twice at 37° C. for 3 days in the NHExpansion Medium (T225 flask). The luciferase expression in MSCs wasconfirmed by luciferase activity assay (Bright-Glo Luciferase AssaySystem, Promega K.K.), and the IL-10 expression was confirmed by ELISAanalysis (canine IL-10 ELISA Kit, Thermo Fisher Scientific Inc.).Appliskan (Thermo Fisher Scientific Inc.) (luminescence, 450 nm, shake)was used in both of the measurements.

2. Allograft Transplantation to Dog and Verification of Survival Rateand Engraftment Rate of Transplanted Cells

An affected dog was prepared as a recipient of the transplanted cells.In order to induce muscle injury in a normal beagle (4 years and 3months old, male), 1.0 mL of 50 μM cardiotoxin was administered by localinjection to each of the right and left tibialis anterior muscles 5 daysbefore transplantation of MSCs to prepare an affected dog. Subsequently,the MSCs introducing each gene in the preceding paragraph “1.Preparation of dog bone marrow-derived MSCs” were recovered by trypsintreatment and then adjusted to 2.4×10⁷ to 2.7×10⁷ cells/2 mL PBS. Under2% isoflurane (Japanese Pharmacopoeia) anesthesia, vIL-10(−) MSCs werelocally transplanted to the left tibialis anterior muscle of theaffected dog, and vIL-10(+)MSCs were locally transplanted to the righttibialis anterior muscle thereof. During this operation, noimmunosuppressant was used. One month later, the MSC-transplanted rightand left tibialis anterior muscle tissues were biopsied and eachsectioned into 4 sites as shown in FIG. 10A. Then, muscle extracts wereobtained using a POLYTRON homogenizer (150 to 180 min⁻¹). Theengraftment of MSCs was analyzed on the basis of the luciferase activity(Bright-Glo Luciferase Assay System) of the muscle extracts. Themeasurement values were corrected with the amount of tissue protein(Pierce® BCA Protein Assay Kit, Thermo Fisher Scientific Inc.). Theamount of IL-10 in the tissue was calculated by ELISA analysis (canineIL-10 ELISA Kit) using the muscle extracts.

After the biopsy of the muscular tissues after the transplantation, asection was prepared as a frozen block from a part of each tissue andpathologically analyzed. MSCs engrafted in the muscular tissue weredetected by DAB staining (VECTASTAIN Elite ABC, Vector Laboratories,Inc.) using an antibody against the marker luciferase introduced in theMSCs (rabbit anti-firefly Luciferase antibody, 1/100 diluted, AbcamPlc.) and subsequent H & E staining in the same way as in Example 2. Thelocalization of the transplanted cells was further observed under amicroscope (Leica, DMR). The tissue was immunohistologically stainedusing rabbit anti-firefly Luciferase antibody (1/50 diluted, Abcam Plc.)and mouse anti-dystrophin antibody (1/100 diluted, NCL-DYS3, Leica), andAlexa 594-conjugated anti-rabbit IgG antibodies (1/250 diluted, LifeTechnologies Corporation) as a secondary antibody, and enclosed in amounting agent Vectashield Mounting Medium with DAPI (VectorLaboratories, Inc.) also serving as nucleic staining. The localizationof the transplanted cells was observed under a fluorescence microscope(Olympus Corp., IX71).

(Results)

The results are shown in FIGS. 9 to 11 .

FIG. 9 shows the image of the engraftment of vIL-10(+)MSCs in the righttibialis anterior muscle tissue, which was the transplantation site, 30days after the transplantation. The DAB-stained dark spots encircled bybroken lines and indicated by arrows represent vIL-10(+)MSCs engraftedin the endomysium and the perimysium.

FIG. 10A is a diagram showing the positions of the 4 sites in theMSC-transplanted right and left tibialis anterior muscle tissues. FIG.10B shows the survival rate of MSCs at each site in the right and lefttibialis anterior muscle tissues and an IL-10 concentration in thetissues 30 days after the transplantation. In the diagram, the survivalrates of vIL-10(−)MSCs (left tibialis anterior muscle tissue) andvIL-10(+)MSCs (right tibialis anterior muscle tissue) based onluciferase activity are shown in (a) and (b), respectively. The IL-10concentration in the muscular tissue of each site in vIL-10(−)MSCs (lefttibialis anterior muscle tissue) and vIL-10(+)MSCs (right tibialisanterior muscle tissue) is shown in (c) and (d), respectively. At theMSC transplantation site TA-2, vIL-10(+)MSCs were shown to have asurvival rate two or more times that of vIL-10(−)MSCs. Thetransplantation of vIL-10(−)MSCs also increased the IL-10 concentrationin the muscular tissue, whereas as a result of transplantingvIL-10(+)MSCs, a high concentration of IL-10 was able to be confirmednot only at the transplantation site but at neighboring sites thereof.No rise was confirmed in IL-10 concentration in serum (not shown). Theseresults are consistent with the results about mice demonstrated inExample 2 and demonstrated that the vIL-10(+)MSCs, which correspond tothe stem cell for transplantation of the present invention, also have ahigh post-transplantation survival rate and engraftment rate in dogs.This suggested that the stem cell for transplantation of the presentinvention is effective regardless of organism species.

FIGS. 11A-B shows the engraftment and myofiber formation ofnon-differentiation-induced MSCs. FIG. 11A shows the pathologic image ofMSCs in the muscular tissue. FIG. 11B shows the ability of MSCs to befused with myotube cells in vitro. As a result of administeringvIL-10(+)MSCs twice to the right tibialis anterior muscle of therecipient dog and conducting long-term observation, the transplantedvIL-10(+)MSCs, as shown in FIG. 11A, were shown to be engrafted at theinflammation site (arrows encircled by broken lines). In FIG. 11A, thearrowheads represent newly formed myofibers. As shown in FIG. 11B, thefusion between MSCs and myoblasts (bright cells encircled by a brokenline) was able to be confirmed in vitro. These results indicate that thestem cell for transplantation of the present invention can be allowed topersistently express IL-10 and engrafted in muscular tissue for a longperiod, thereby newly reforming myofibers through the fusion withneighboring myoblasts. Adverse events such as hematopoietic injury ortumor formation were not observed.

Example 4 <Verification of Induction of Acquired Immunological ToleranceUsing MSCs-(1)>

(Objective)

The objective is to verify, in dogs, that immunological toleranceagainst immunogen to be introduced to recipient individual is induced bypretreatment using MSCs.

(Method)

The basic method followed the method described in Example 3.

1. Immunological Tolerance Induction Procedures

On the basis of the method for inducing immunological tolerance of thepresent invention, the immunological tolerance was induced by thefollowing procedures: first, AAV9 was introduced as an immunogen to eachrecipient, and this administration date was defined as a reference date(day 0). Next, 8 days before the reference date, MSCs and the immunogenAAV9 were intravenously administered as the first step of the method forinducing immunological tolerance. For a control, only AAV9 or only MSCswere intravenously administered. Subsequently, on the day before thereference date, MSCs were intravenously administered as the second stepof the method for inducing immunological tolerance. This administrationwas not carried out for the control. On the reference date, theimmunogen AAV9 was introduced by local administration. The introducedAAV9 causes luciferase gene expression. Only an AAV9-free PBS buffer wasintroduced to the control. 4 weeks after the introduction, biopsy wasconducted.

2. Preparation of Dog Bone Marrow-Derived MSCs

The basic method followed the method described in Example 3.

3. Preparation of AAV9-Luc

The luciferase expression AAV vector (AAV9-CAG-Luc: in the presentspecification, also referred to as “AAV9-Luc”) introduced andadministered as an immunogen in this Example was prepared on the basisof the method described in the paragraph “1. Preparation of recombinantAAV” of Comparative Example 2.

The AAV9-Luc was prepared according to the method described in OhshimaS., et al., 2009, Mol Ther, 17 (1): 73-80 or Shin J. H., et al., 2011,Gene Ther, 18: 910-919. The AAV9-Luc is capable of persistentlyexpressing luciferase in a tissue that has received it.

4. Verification of Induction of Immunological Tolerance by LocalAdministration

The recipients used were normal beagles (2-month-old male or femalelittermates). According to the preceding paragraph “1. Immunologicaltolerance induction procedures”, 8 days before the reference date, 4×10⁶cells (1×10⁶ cells/kg B.W.) of MSCs and 2 mL of an AAV9-Luc solutionwere locally administered using a syringe to the tibialis anteriormuscle of each dog (MSCs+AAV) for immunological tolerance induction. Fora control, only AAV9-Luc (Cont-AAV) or only MSCs (Cont-MSCs) wereadministered. On the day before the reference date, MSCs wereadministered at 1.8×10⁶ cells/kg B.W. to the individual MSCs+AAV orCont-MSCs. On the reference date, introduction to the tibialis anteriormuscle was carried out using a syringe such that AAV9-Luc was locallyadministered at 1×10¹² v.g. (solution volume: 2 mL) per site to twosites in the individual MSCs+AAV or Cont-AAV, and 2 mL of a PBS bufferalone was locally administered to each of two sites in the individualCont-MSCs. 4 weeks after the reference date, the tibialis anteriormuscle was biopsied at the introduction sites. The tissue wasimmunohistologically stained using an antibody against luciferase(rabbit anti-firefly Luciferase antibody, 1:2000 diluted, Abcam Plc.)and Alexa 594-conjugated anti-rabbit IgG antibodies (1:1000 diluted,Life Technologies Corporation) as a secondary antibody. The expressionof luciferase was observed under a fluorescence microscope (OlympusCorp., IX71).

(Results)

The results are shown in FIGS. 12A-C. FIG. 12A shows the results aboutCont-AAV. FIG. 12B shows the results about MSCs+AAV. FIG. 12C shows theresults about Cont-MSCs. In the MSCs+AAV treated by the method forinducing immunological tolerance of the present invention, theexpression of the marker gene of luciferase derived from the immunogenAAV9 was observed (FIG. 12B). By contrast, in the Cont-AAV pretreatedwith only AAV9 without the immunological tolerance induction treatmentwith MSCs, the expression of luciferase was not seen (FIG. 12A).Likewise, in the Cont-MSCs that underwent neither administration norintroduction of AAV9-Luc, the expression of luciferase was not seen(FIG. 12C).

These results indicate that immunological tolerance against theimmunogen AAV9-Luc was induced in the individual that received theimmunological induction treatment by the method of the presentinvention, and the introduced AAV9-Luc was not eradicated by immuneresponse even 4 weeks after the introduction of the AAV9-Luc.

Example 5 <Verification of Induction of Acquired Immunological ToleranceUsing MSCs-(2)>

(Objective)

The objective is to verify that in the introduction of an immunogenaccording to the method for inducing acquired immunological tolerance ofthe present invention, even intravenous introduction produces effectssimilar to those of local introduction.

(Method)

1. Immunological tolerance induction procedures

The basic procedures followed the procedures of Example 4. In thisExample, AAV9-Luc or AAV-μDys was introduced as an immunogen.

2. Human-Derived MSCs

Human-derived bone marrow-derived cells (JCR Pharmaceuticals Co., Ltd.)were purchased and then expanded by culture for use.

3. Preparation of AAV9-μDys

The AAV9-μDys to be introduced and administered was prepared on thebasis of the method described in the paragraph “1. Preparation ofrecombinant AAV” of Comparative Example 2.

The microdystrophin expression AAV vector (AAV9-CMV-μDys; in the presentspecification, also referred to as “AAV9-μDys”) was prepared accordingto the AAV9-Luc preparation method described in the paragraph 3 ofExample 4. Microdystrophin is a truncated form of the dystrophin genedeveloped so as to permit integration into an AAV vector, and has thesame functions as those of the dystrophin gene. The AAV9-μDys is capableof persistently expressing microdystrophin in a tissue that has receivedit.

4. Verification of Induction of Immunological Tolerance by IntravenousIntroduction

The recipients used were two 11-week-old littermates of normal beagles(male) or one Duchenne dystrophy-affected dog (male) (CXMD_(J)). TheCXMD_(J) is a hybrid that has been bred and maintained by crossing thesperm of a golden retriever spontaneous Duchenne dystrophy-affected dog(GRMD) imported from abroad with a beagle in the National Center ofNeurology and Psychiatry (Japan) (Shimatsu Y. et al., 2005, Acta Myolvol. 24 (2): 145-54; Yugeta N. et al., 2006, BMC Cardiovasc Disord, 6:47; and Kobayashi M et al., 2009, Muscle Nerve, 40 (5): 815-26).

According to the preceding paragraph “1. Immunological toleranceinduction procedures”, 8 days before the reference date, 1×10⁶ cells/kgB.W. of MSCs and 3 mL of AAV9-Luc were intravenously injected to eachnormal beagle (MSCs+AAV-Luc), and 1×10⁶ cells/kg B.W. of MSCs and 3 mLof AAV9-μDys were intravenously injected to the affected dog(DMD/MSCs+AAV-μDys), for immunological tolerance induction. For acontrol, only AAV9-Luc (Cont-AAV) was administered. Each amount of AAVwas set to 5×10⁵ v.g./cell. On the day before the reference date, 2×10⁶cells/kg B.W. of MSCs were administered to the individual MSCs+AAV-Lucor DMD/MSCs+AAV-μDys. On the reference date, 2×10¹² v.g./kg B.W. ofAAV9-Luc was intravenously introduced to the individual MSCs+AAV-Luc orCont-AAV, and 2×10¹² v.g./kg B.W. of AAV9-μDys was intravenouslyintroduced to the DMD/MSCs+AAV-μDys (solution volume: 3 mL each). 4weeks after the reference date, the temporal muscle was excised fromeach dog and biopsied. The tissue was immunohistologically stained usingan antibody against luciferase (rabbit anti-firefly Luciferase antibody,1:2000 diluted, Abcam Plc.) and an antibody against dystrophin (mouseanti-dystrophin antibody, 1:50 diluted, NCL-DYS3, Leica), and Alexa594-conjugated anti-rabbit IgG antibodies (1:1000 diluted, LifeTechnologies Corporation) as a secondary antibody, and enclosed in amounting agent Vectashield Mounting Medium with DAPI (VectorLaboratories, Inc.) also serving as nucleic staining. The expression ofluciferase or microdystrophin was observed under a fluorescencemicroscope (Olympus Corp., IX71).

(Results)

The results are shown in FIGS. 13A-C. FIG. 13A shows the results aboutCont-AAV. FIG. 13B shows the results about MSCs+AAV-Luc. FIG. 13C showsthe results about DMD/MSCs+AAV-μDys. In the MSCs+AAV-Luc treated by themethod for inducing immunological tolerance of the present invention,the expression of luciferase was observed (FIG. 13B), as in Example 4.By contrast, in the Cont-AAV pretreated with only AAV9 without theimmunological tolerance induction treatment with MSCs, the expression ofluciferase was not seen (FIG. 13A). These results demonstrated that inthe introduction of an immunogen by immunological tolerance inductiontreatment, not only local introduction but systemic administrationmediated by the circulatory system, such as intravenous introduction,produces similar effects.

In the DMD/MSCs+AAV-μDys, the expression of microdystrophin wasconfirmed (FIG. 13C), as in the MSCs+AAV-Luc given luciferase. Theseresults indicate that immunological tolerance against the immunogenAAV9-μDys was induced, and the introduced AAV9-μDys was not eradicatedby immune response even 4 weeks after the introduction of the AAV9-μDys.

Example 6 <Verification of Gene Therapy by Method for Inducing AcquiredImmunological Tolerance>

(Objective)

The objective is to verify the effect of gene therapy on anAAV9-μDys-introduced individual using the method for inducing acquiredimmunological tolerance of the present invention.

(Method)

1. Preparation of Various Dogs

11-week-old littermates of normal beagles (Normal), musculardystrophy-affected dogs (DMD), and treated affected dogs(DMD/MSCs+AAV-μDys) in which AAV9-μDys was introduced to the affecteddogs using the method for inducing acquired immunological tolerance ofthe present invention, were prepared. The DMD is the aforementionedCXMD_(J) affected dog. The DMD/MSCs+AAV-μDys is the AAV9-μDys-introducedaffected dog prepared in Example 5.

2. Verification of Effect of Gene Therapy-(1)

A 15-meter running test was conducted using each dog, and its runningtime was measured. As for DMD, only one measurement was carried out for5 individuals at each weekly age. As for Normal and DMD/MSCs+AAV-μDys, 4measurements were carried out per individual, and an average timethereof was calculated.

3. Verification of Effect of Gene Therapy-(2)

The pathological evaluation of muscular dystrophy followed the GradingScore (ver. 10) used in the National Center of Neurology and Psychiatry(Japan). In this Grading Score, regarding muscular dystrophy-relatedpathological conditions of 14 items [eating/swallowing, drinking,salivation, dysphagia, abnormal phonation, test of getting up from alying position, playfulness, gait, hindlimb gaiting test, seatedposture, temporal muscle atrophy, sublingual swelling, macroglossia, andthigh muscle], appropriate statuses were selected from those specifiedin each item, and scores assigned in advance to the statuses were summedup for scoring. A larger score means more severe symptoms. Thepathological evaluation was conducted once a month.

(Results)

The results of the 15-meter running test are shown in FIG. 14 . Changein Grading Score as the pathological evaluation of muscular dystrophy isshown in FIG. 15 .

As seen from FIG. 14 , the gene therapy-untreated musculardystrophy-affected dogs (DMD) had a slower running time than that of thenormal individuals (Nomal) at every weekly age, showing motordysfunction. By contrast, the muscular dystrophy-treated affected dogs(DMD/MSCs+AAV-μDys) subjected to the immunological tolerance inductiontreatment and given AAV9-μDys using the method for inducing acquiredimmunological tolerance of the present invention had a running timecloser to that of the normal individuals, as compared with the runningtime of DMD. This effect was also maintained even 17 weeks after theintroduction of AAV9-μDys.

The results of the pathological evaluation of muscular dystrophy areshown in FIG. 15 . As seen from FIG. 15 , the symptoms of the genetherapy-untreated muscular dystrophy-affected dogs (DMD1 and DMD2)progressed and became more severe with increases in monthly age. Bycontrast, the symptoms of the muscular dystrophy-treated affected dogs(DMD/MSCs+AAV-μDys) given AAV9-μDys using the method for inducingacquired immunological tolerance of the present invention progressedvery slowly even with increases in monthly age, as compared with DMD.

These results indicate that the treatment of a recipient by the methodfor inducing acquired immunological tolerance of the present inventionallows gene therapy to maintain its effects over long period withouteradicating the introduced AAV9-μDys by immune response.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

1-11. (canceled)
 12. A composition comprising human mesenchymal stemcells (hMSCs) and a viral immunogen, wherein, when the composition isadministered to a subject, it suppresses the subject's immune responseto the viral immunogen.
 13. The composition according to claim 12,wherein the viral immunogen is from an adenovirus, adeno-associatedvirus (AAV), retrovirus, or lentivirus.
 14. The composition according toclaim 13, wherein the viral immunogen is from an AAV or adenovirus. 15.The composition according to claim 12, wherein the viral immunogen is arecombinant viral immunogen.
 16. The composition according to claim 15,wherein the recombinant viral immunogen is a recombinant virus forexpression of IL-10.
 17. The composition according to claim 12, whereinthe composition induces tolerance to the viral immunogen whenadministered to the subject.
 18. The composition according to claim 12,wherein the hMSCs are recombinant hMSCs.
 19. The composition accordingto claim 18, wherein the recombinant hMSCs overexpress exogenous IL-10.20. The composition according to claim 12, further comprisingrecombinant IL-10.
 21. A method for reducing an immune response to aviral immunogen in a subject in need thereof, the method comprising:administering to the subject: (i) human mesenchymal stem cells (hMSCs);and (ii) the viral immunogen, whereby the immune response to the viralimmunogen is suppressed.
 22. The method according to claim 21, whereinthe viral immunogen is from an adenovirus, adeno-associated virus (AAV),retrovirus, or lentivirus.
 23. The method according to claim 21, whereinthe viral immunogen is a recombinant viral immunogen.
 24. The methodaccording to claim 23, wherein the recombinant viral immunogen is arecombinant virus for expression of IL-10.
 25. The method according toclaim 22, wherein the viral immunogen is from an AAV or adenovirus. 26.The method according to claim 21, wherein suppression of the immuneresponse comprises induction of tolerance to the viral immunogen. 27.The method according to claim 21, wherein the hMSCs are recombinanthMSCs.
 28. The method according to claim 27, wherein the recombinanthMSCs overexpress exogenous IL-10.
 29. The method according to claim 21,wherein the hMSCs and the viral immunogen are co-administered at thesame time.
 30. The method according to claim 21, wherein the hMSCs andthe viral immunogen are administered sequentially.
 31. The methodaccording to claim 21, further comprising administering recombinantIL-10.